Biomedical subjects

Lumelle A Schneeweis

Publications and source records attributed to Lumelle A Schneeweis.

3 recordsLinked to original sources

PubMed2005

Functional dissection of osteoprotegerin and its interaction with receptor activator of NF-kappaB ligand.

The receptor activator of NF-kappaB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nM. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of approximately 3 microM, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction.

Animals↗

PubMed2005

Structural analysis of lipoprotein E particles.

Apolipoprotein E (apoE) is a key regulator of cholesterol homeostasis. Human apoE has three common isoforms, each with different risk implications for cardiovascular and neurodegenerative disease. Neither the structure of lipoprotein E particles nor the structural consequences of the isoform differences are known. In this investigation, synthetic lipoprotein particles were prepared by complexing phospholipids with full-length apoE isoforms, or with truncated N-terminal and C-terminal domains of apoE. These particles were examined with calorimetry, electron microscopy, circular dichroism spectroscopy, and internal reflection infrared spectroscopy. Results indicate that particles made with the three full-length apoE isoforms are discoidal in shape, and structurally indistinguishable. Thus, differences in their pathological consequences are not due to gross differences in particle structure. Although apoE is predominantly helical, and the axes of the helices are parallel to the flat surfaces of the particles, the orientational order of lipid acyl chains is low and inconsistent with the belt model of lipoprotein A-I structure. Instead, the data suggest that there are at least two different types of apoE-lipid interactions within lipoprotein E particles. One type occurs between apoE helices and the edge of the lipid bilayer as in the belt model, while a second type involves apoE helices that situate in the plane of the membrane and disturb acyl chain order. These interactions allow LpE particles to form with different protein/lipid ratios, and they account for the structure of LpE particles made with only the truncated domains.

Apolipoproteins E↗

PubMed2003

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

Animals↗